T7 RNA polymerase (E.C. 2.7.7.6.), also referred to herein as “T7 polymerase” or “T7,” is a monomeric bacteriophage encoded DNA directed RNA polymerase which catalyzes the formation of RNA in the 5′→3′ direction. In the process of initiation of transcription, T7 recognizes a specific promoter sequence, the T7 promoter. T7 consists of 883 amino acids and has a molecular weight of 99 kDa. At the amino acid sequence level, T7 is highly homologous to T3 RNA polymerase and, to a lesser extent, SP6 RNA polymerase. The three-dimensional structure of T7 is also similar to other polymerases with different template and substrate specificities, despite low sequence similarity. T7 consists of different domains, the N-terminal domain, the “thumb”, the “palm” and the “fingers” (as described in Sousa, R., and Mukherjee, S., Prog. Nucl. Acid Res. Mol. Biol. 73 (2003) 1-41).
Cloning and expression of the gene encoding T7 has been described in literature (see, U.S. Pat. No. 4,952,496, the disclosure of which is expressly incorporated by reference). Conformational changes of T7 during transcription (see, Ma, K., et. al., Proc. Nat. Acad. Sci. 102 (2005) 17612-17617), the facilitation of promoter clearance of T7 (see, Guillerez, J., et al., Proc. Natl. Acad. Sci. 102 (2005) 5958-5963) and the abortive cycling phenomenon of T7 (see, He, B., et al., J. Mol. Biol. 265 (1997) 275-288) have all been studied.
T7 has been classified as having high promoter specificity and RNA polymerase enzymatic activity, and as being useful for a variety of applications in molecular biology. In the field of recombinant protein expression, T7 is used for the high-level expression of recombinant genes in E. coli (for example, as discussed in Studier, F. W., and Moffat, B. A., J. Mol. Biol. 189 (1986) 113-130). The synthesis of defined oligoribonucleotides was described by Milligan, J. F., et al., Nucl. Aids Res. 15 (1987) 8783-8798. However, for at least some applications with which T7 RNA Polymerase may be utilized a more stable T7 RNA polymerase would be of value.